chlorophyll-a and Melanoma--Amelanotic

Pheophorbide a (Pba) is a chlorophyll catabolite that has been proposed as photosensitizer in photodynamic therapy. In a previous study we conjugated Pba to monomethoxy-polyethylene glycol (mPEG-Pba), to increase its solubility and pharmacokinetics. Here, we compare the photodynamic therapy efficacy of free Pba and mPEG-Pba to cure a subcutaneous amelanotic melanoma transplanted in C57/BL6 mice. The photosensitizers, i.p. injected (30 mg/kg), showed no toxicity when the animals were kept in the dark. But, after photoactivation with a 660 nm laser (fluence of 193 J/cm(2)), both photosensitizers, in particular mPEG-Pba, showed a strong efficacy to cure the tumor, both in terms of tumor growth delay and increase of Kaplan-Meier median survival time. Together, our in vivo data demonstrate that mPEG-conjugated Pba is a promising photosensitizer for the photodynamic therapy of cancer.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Chlorophyll; Female; Light; Melanoma, Amelanotic; Mice; Mice, Inbred C57BL; Photochemotherapy; Polyethylene Glycols; Radiation-Sensitizing Agents

Cell recurrence in cancer photodynamic therapy (PDT) is an important issue that is poorly understood. It is becoming clear that nitric oxide (NO) is a modulator of PDT. By acting on the NF-κB/Snail/RKIP survival/anti-apoptotic loop, NO can either stimulate or inhibit apoptosis. We found that pheophorbide a/PDT (Pba/PDT) induces the release of NO in B78-H1 murine amelanotic melanoma cells in a concentration-dependent manner. Low-dose PDT induces low NO levels by stimulating the anti-apoptotic nature of the above loop, whereas high-dose PDT stimulates high NO levels inhibiting the loop and activating apoptosis. When B78-H1 cells are treated with low-dose Pba/PDT and DETA/NO, an NO-donor, intracellular NO increases and cell growth is inhibited according to scratch-wound and clonogenic assays. Western blot analyses showed that the combined treatment reduces the expression of the anti-apoptotic NF-κB and Snail gene products and increases the expression of the pro-apoptotic RKIP gene product. The combined effect of Pba and DETA/NO was also tested in C57BL/6 mice bearing a syngeneic B78-H1 melanoma. We used pegylated Pba (mPEG-Pba) due to its better pharmacokinetics compared to free Pba. mPEG-Pba (30 mg/Kg) and DETA/NO (0.4 mg/Kg) were i.p. injected either as a single molecule or in combination. After photoactivation at 660 nM (fluence of 193 J/cm(2)), the combined treatment delays tumor growth more efficiently than each individual treatment (p<0.05). Taken together, our results showed that the efficacy of PDT is strengthened when the photosensitizer is used in combination with an NO donor.

Topics: Animals; Cell Line, Tumor; Chlorophyll; Female; Flow Cytometry; Melanoma, Amelanotic; Mice; Mice, Inbred C57BL; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Photochemotherapy; Photosensitizing Agents; Skin Neoplasms; Wound Healing

Photodynamic therapy (PDT) is a therapeutic modality whose efficacy depends on several factors including type of photosensitizer, light fluence and cellular response. Cell recurrence is one of the problems still unsolved in PDT. In this work we found that in B78-H1 murine amelanotic melanoma cells there is a correlation between cell recurrence and the NF-κB/Snail/RKIP loop.. Proliferation and migration of surviving cells were analyzed by MTT and wound-scratch assays. The levels of ROS/NO in B78-H1 melanoma cells treated with pheophorbide a (Pba) and light (Pba/PDT) were measured by FACS, while expression of NF-κB, Snail and RKIP were determined by Western blots. The mechanism of cell death was investigated by caspase and microscopy assays.. Our data show that after a low-dose Pba/PDT treatment, B78-H1 cells are able to recover. This correlates with a low level of NO production, which blocks apoptosis via NF-κB pathway. Western blot analyses showed that a low-dose Pba/PDT increases the expression of NF-κB and anti-apoptotic Snail, but reduces the expression of pro-apoptotic RKIP. The role played by NF-κB in the modulation of Snail and RKIP was investigated using DHMEQ: a NF-κB inhibitor which behaves as NO donor. DHMEQ caused a decrease of Snail and an increase of RKIP expression. When B78-H1 cells were treated with a low dose Pba/PDT and DHMEQ, the NO level strongly increased, with the result that Snail was down-regulated and RKIP was upregulated, as observed with a high-dose Pba/PDT.. One major problem in PDT is the cellular rescue occurring in tissue regions receiving a low-dose PDT. To minimize this problem and sensitize cancer cells to PDT we propose a combined treatment in which the photosensitizer is delivered with a donor of NO acting on the NF-κB/Snail/RKIP loop.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chlorophyll; Dose-Response Relationship, Drug; Melanoma, Amelanotic; Mice; NF-kappa B; Nitric Oxide; Phosphatidylethanolamine Binding Protein; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species; Skin Neoplasms; Snail Family Transcription Factors; Transcription Factors